Human Endogenous Retrovirus-K(II) Envelope Induction Protects Neurons during HIV/AIDS

<div><p>Human endogenous retroviruses (HERVs) are differentially expressed depending on the cell type and physiological circumstances. HERV-K has been implicated in the pathogenesis of several diseases although the functional consequences of its expression remain unknown. Human immunodeficiency virus (HIV) infection causes neuroinflammation with neuronal damage and death. Herein, we investigated HERV-K(II)/(HML-2) envelope (Env) expression and its actions in the brain during HIV/AIDS. HERV-K(II) Env expression was assessed in healthy brain tissues, autopsied HIV HIV− infected (HIV+) and uninfected (HIV−) brains and in neural cell cultures by real time RT-PCR, massively parallel (deep) sequencing, immunoblotting and immunohistochemistry. Neuronal and neural stem cells expressing HERV-K(II) Env were analyzed in assays of host responses including cellular viability, immune responses and neurobehavioral outcomes. Deep sequencing of human brain transcriptomes disclosed that RNA sequences encoded by HERV-K were among the most abundant HERV sequences detected in human brain. Comparison of different cell types revealed that HERV-K(II) <i>env</i> RNA abundance was highest in cultured human neurons but was suppressed by epidermal growth factor exposure. HERV-K(II) Env immunoreactivity was increased in the cerebral cortex from persons with HIV/AIDS, principally localized in neurons. Human neuronal cells transfected with HERV-K(II) Env exhibited increased <i>NGF</i> and <i>BDNF</i> expression. Expression of HERV-K(II) Env in neuronal cells increased cellular viability and prevented neurotoxicity mediated by HIV-1 Vpr. Intracerebral delivery of HERV-K(II) Env expressed by neural stem cells suppressed TNF-α expression and microglial activation while also improving neurobehavioral deficits in <i>vpr/RAG1^−/−</i> mice. HERV-K(II) Env was highly expressed in human neurons, especially during HIV/AIDS, but in addition exerted neuroprotective effects. These findings imply that HERV gene products might exert adaptive effects in circumstances of pathophysiological stress, perhaps underlying the conservation of HERVs within the human genome.</p></div

Linking climate warming and land conversion to species’ range changes across Great Britain

Although increased temperatures are known to reinforce the effects of habitat destruction at local to landscape scales, evidence of their additive or interactive effects is limited, particularly over larger spatial extents and longer timescales. To address these deficiencies, we created a dataset of land-use changes over 75 years, documenting the loss of over half (>3000 km2) the semi-natural grassland of Great Britain. Pairing this dataset with climate change data, we tested for relationships to distribution changes in birds, butterflies, macromoths, and plants (n = 1192 species total). We show that individual or additive effects of climate warming and land conversion unambiguously increased persistence probability for 40% of species, and decreased it for 12%, and these effects were reflected in both range contractions and expansions. Interactive effects were relatively rare, being detected in less than 1 in 5 species, and their overall effect on extinction risk was often weak. Such individualistic responses emphasise the importance of including species-level information in policies targeting biodiversity and climate adaptation

HERV transcripts in HIV− infected brain specimens.

<p>(<b>A</b>) Deep sequencing of the HIV− and HIV+ autopsied cerebral white matter revealed a higher tag frequency of HERV-K in both clinical groups compared to other HERVs. (<b>B</b>) With the use of the DAVID bioinformatics resources, the predicted target genes were classified according to KEGG functional annotations to identify pathways that were actively regulated by HERV-K(II) <i>env</i> transcripts in brain tissue.</p

Over expression of HERV-K(II) Env exerts neurotrophic effects:

<p>(<b>A</b>) Transfection of the pHERV-Kenv plasmid into SK-N-SH cells showed HERV-K(II) Env immunoreactivity at the predicted molecular weight on western blot. (<b>B</b>) Upon treatment with supernatants from SK-N-SH cells transfected with pHERV-Kenv plasmid, HFN showed increases in <i>BDNF</i> and <i>NGF</i> transcript abundance compared to the control vector transfected cells. (n = 3, with technical quadruplicates) (<b>C</b>) βIII-tubulin expression in HFN following 24-hour exposure to supernatants from HFA-transfected with the pHERV-Kenv or the control vector, showing an increase in βIII-tubulin immunoreactivity in cells exposed to HERV-K Env-transfected cells. (n = 2, with technical octuplicates) (Student t test, *<i>p</i><0.05, **<i>p</i><0.01).</p

Deep sequencing analyses of HERVs in healthy brain.

<p>(<b>A</b>) Deep sequencing of the fetal and surgically resected (Surg) brain samples revealed that HERV-H exhibited the highest tag frequency and median number of tags followed by HERV-K. (<b>B</b>) When analyzing the HERV-K tags, LTR tags were most abundant, followed by <i>gag-pol</i> and then the <i>env</i> region tags (tags were normalized to respective gene lengths) (<b>C</b>) All host genes with transcript expression profiles correlated with HERV-K(II) <i>env</i> tag abundance (r²<b>≥</b>0.5) were analyzed using the DAVID tools <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097984#pone.0097984-DAVID1" target="_blank">[58]</a> for enriched gene ontology (GO) terms. Genes related to cell cycle functions and chromosomal organization were most strongly associated with HERV-K(II) <i>env</i> expression. With the use of DAVID bioinformatics resources <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097984#pone.0097984-Dennis1" target="_blank">[59]</a>, the predicted target genes were classified according to KEGG functional annotations to identify pathways that were actively regulated by HERV-K(II) <i>env</i> transcripts in brain tissue. The most over-represented GO term belonged to the transcriptional regulation and chromosome organization followed by different stages of cell cycle pathway. (Mann Whitney t test, *<i>p</i><0.05, **<i>p</i><0.01).</p

Oligonucleotide primers used in Real-time RT PCR analyses.

<p>Oligonucleotide primers used in Real-time RT PCR analyses.</p

Activation of HERV-K(II) <i>env</i> by cAMP and EGF in different human cell lines

<p>(<b>A</b>) Individual cell lines displayed differential constitutive HERV-K(II) <i>env</i> expression profiles. (<b>B</b>) Upon treatment of human fetal neurons, db-cAMP did not have any effects on HERV-K(II) <i>env</i> expression but EGF down-regulated HERV-K(II) expression. (<b>C</b>) U937 and (<b>D</b>) HFA showed decreased in HERV-K(II) <i>env</i> expression upon both db-cAMP and EGF exposure. (n = 4 replicates per group across two independent experiments).</p

HERV-K(II) <i>env</i> transfection of neuronal cells was neuroprotective.

<p>(<b>A</b>) Analyses of SK-N-SH cells transfected with the pHERV-Kenv plasmid compared to the control (pGFP) showed that the efficiency of transfection was ∼20% (n = 3, with technical triplicates). (<b>B</b>) HERV-K(II) Env immunoreactivity was minimally detected in cells transfected with the control vector. pHERV-Kenv-transfected cells showed HERV-K(II) Env immunoreactivity at low (<b>C</b>) and high magnification (<b>D</b>). (<b>E</b>) Comparison of <i>BDNF</i> and <i>NGF</i> transcript levels in SKN-N-SH cells transfected with pGFP or pHERV-Kenv. (<b>F</b>) Exposure of pHERV-Kenv and control vector-transfected NG108 cells to staurosporine, HIV-1 Vpr or NMDA, showed that pHERV-Kenv-transfected cells were differentially protected depending on the neurotoxin. (Student t test, *<i>p</i><0.05, ***<i>p</i><0.001).</p

Neural stem cells expressing HERV-K(II) Env are protective in <i>vpr/RAG1^−/−</i> animals.

<p>(<b>A</b>) Schematic of representation of C17.2 implantation site (marked by the •) in Vpr/RAG1^−/− mice. (<b>B</b>) Western blot showing HERV-K(II) Env immunoreactivity in transfected cells. (<b>C</b>) <i>TNF-α</i> expression was suppressed in the brains of animals implanted with cells expressing HERV-K(II) <i>env</i> while (<b>C</b>) <i>IL-6</i> was induced. Nissl staining showed similar striatal neuronal densities in animals implanted with cells transfected with either pGFP or pHERV-Kenv/pGFP (<b>E, I</b>). Immunohistochemistry revealed lower expression the microglia protein, Iba-1 (<b>K</b>) <b>and</b> higher expression levels astrocyte protein, GFAP (<b>L</b>) in HERV-K(II) <i>env</i> implanted brains compared to control vector (<i>pGFP</i>) implanted animals (<b>F, G</b>), respectively. Cleaved caspase-6 immunoreactvity was comparative reduced in striatum of animals receiving cells transfected with pHERV-Kenv/pGFP (<b>M</b>) but BDNF immunoreactivity was increased in the same animals (<b>N</b>) compared to controls (<b>H, I</b>). (<b>O</b>) At days 7 and 14, neurobehavioral deficits were greater in terms of ipsiversive rotations among the animals implanted with c17.2 cells transfected with the <i>pGFP</i> vector. (Original magnification: E–J, 400X) (Mann-Whitney test, *<i>p</i><0.05).</p